Gottesman Project Title Summary We have focused our studies on two types of post-transcriptional regulation of gene expression, regulation of mRNA degradation and translation by small regulatory RNAs and regulation of protein stability by energy-dependent proteases. RpoS, a central stress response regulator in Escherichia coli, is subject to both of these levels of control. Degradation of RpoS requires ClpXP protease, and RssB, a protein that presents RpoS to the protease. Degradation is signaled by phosphorylation of RssB; we are investigating the mechanism of phosphorylation and dephosphorylation in vivo and in vitro. However, recent studies demonstrated significant regulation of degradation in the absence of phosphorylation, suggesting a novel method of regulation that needs to be understood. Deletion analysis of RssB indicates that sequences at the C-terminus are critical for proper release of the substrate to the protease; N-terminal sequences are required for interaction with the protease. RpoS translation is positively regulated by at least two small RNAs. The message upstream of the RpoS translation start folds into a hairpin that occludes ribosome binding and therefore translation. The small regulatory RNAs, DsrA and RprA, compete for the inhibitory stem of the hairpin, disrupting the secondary structure of the RpoS leader, allowing translation. The promoter of dsrAis regulated by temperature ; we have previously shown that regulation resides in a minimal promoter region, with the -10 region playing a critical role. RprA, identified as a multicopy suppressor of dsrAmutants, is regulated by the RcsC, RcsD, RcsB phosphorelay regulatory system. These regulators also act to turn up capsular polysaccharide synthesis and to up regulate a cell division protein as well as many other genes, some involved in biofilm formation; they are activated by cell surface stress. Genetic studies have demonstrated that sensing to activate the phosphorelay is usually via modification of the activity or structure of RcsF, a lipoprotein that acts upstream of the membrane sensor, RcsC. Two additional small RNAs that appear to regulate RpoS are under study. Finally, some novel levels of regulation of translation of RpoS have been found that are independent of small RNAs. Both tmRNA, an RNA that is used at stalled ribosomes to tag the peptide for degradation and relieve the stalling, and the miaA-directed RNA modification are necessary for high levels of RpoS translation. Thus, a variety of cellular systems affect RpoS levels. A genome-wide search for conserved small RNAs was carried out in collaboration with Gisela Storz's laboratory in NICHD. We have investigated the roles and regulation for a number of the small RNAs found in that search. The best-studied of these is RyhB. RyhB transcription is repressed by the Fur, iron-dependent repressor, and the small RNA is therefore made in high quantities when intracellular iron is limiting. When it is made, it targets multiple mRNAs for degradation. The target mRNAs encode either iron storage proteins (ferritins) or iron-containing but non-essential metabolic proteins. Therefore, this small RNA, which is also found in Vibrio, Salmonella, Klebsiella, and Yersinia, reprograms iron use in the cells and may be an important component of virulence for some pathogens. Using the properties of RyhB, we collaborated with Dr. David Fitzgerald and Dr. Peter Fitzgerald at NCI as well as Dr. Michael Vasil in Colorado to demonstrate that similar small RNAs regulate genes encoding iron-containing proteins in Pseudomonas aeruginosa, although the sequences of the regulatory RNAs are very different. In further studies on RyhB, we demonstrated that both RyhB and its target mRNAs are rapidly degraded, dependent upon an essential endonuclease, RNase E. Degradation of the small RNA was only rapid when transcription of other genes was active, suggesting that pairing with mRNA targets was critical for degradation of the small RNA. Based on the studies of RyhB, we were able to show that two other small RNAs of this class, DsrA and OxyS, were also unstable when transcription was active but stable when it was inactive; this is probably a general property of these small RNAs. Two other small RNAs from this search, RygA and RygB, have been found to regulate a number of outer membrane proteins; these small RNAs are made after osmotic shock and appear to be part of the OmpR/EnvZ regulon. In a continued collaboration with Dr. Gisela Storz's laboratory, we used the ability of the RNA chaperone Hfq to bind tightly to many small RNAs to identify yet more small RNAs in E. coli. Hfq was immunoprecipitated, the bound RNA isolated and used to probe microarrays. At least six additional small RNAs were identified with another 6-10 likely; all use Hfq, which suggests that they act by pairing with target mRNAs. One of these, originally called RyaA and now renamed SgrS, has been studied in some detail. SgrS is made when cells accumulate glucose-6-phosphate or a phosphorylated glucose analog. The sugar phosphate is made when glucose is transported into the cells by the glucose PTS transporter; the glucose-specific gene is encoded by ptsG. Work by H. Aiba and colleagues had demonstrated that when further metabolism is blocked, the accumulation of the sugar phosphate is toxic and cells respond by degrading the mRNA for ptsG. We find that this is mediated by SgrS, which is predicted to pair with the ptsG 5' UTR. SgrS induction depends on a novel transcriptional regulator, encoded by the divergent gene, yabN , renamed by us sgrR . The SgrR protein may directly sense the accumulation of sugar phosphate. When either the small RNA or the transcriptional regulator are mutant, cells are unable to recover from glucose-phosphate accumulation. The action of DsrA and RprA in positive regulation of RpoS translation is rather unique and raised the question whether pairing simply opens a hairpin or has effects on the processing or stability of the target mRNA and the small RNA. Using previously tested mutations and compensating mutations in the small RNAs and the target messages to provide specificity, we can show that small RNA pairing with the rpoS message results in higher levels of the message, and also seems to stabilize the small RNAs. Further studies on this should increase our understanding of positive action of small RNAs.